ABSTRACT Intracellular calcium signals play a vital role in regulating immune system homeostasis and function. In T cells, calcium ions serve as a critical second messenger in a broad variety of cellular processes regulating T cell activation, proliferation and differentiation of nave T cells into effector or memory cells. The mechanism supporting the sustained calcium influx into the cytoplasm is known as store-operated calcium entry (SOCE). Two proteins, Orai1 and STIM1, were identified as primary modulators of SOCE in T cells. SOCE is initiated when STIM1 senses the depletion of internal calcium stores and associates with the pore-forming Orai1 to assemble the calcium release-activated channel. The critical role of STIM1 and Orai1 in the regulation of T cell immune responses is well supported by genetic studies performed in animals as well as clinical data. Biological consequences of Orai1 or STIM1 deficiencies include severe immunodeficiency, tubular aggregate myopathy, and Stormorken syndrome. In our preliminary experiments, we have identified both Orai1 and STIM1 as endogenously S-acylated proteins. S-acylation, a reversible post-translational lipidation of cysteine residues with long-chain fatty acids, is catalyzed by the family of DHHC palmitoyl acyltransferases known to regulate the function of many key T cell signaling proteins. Our previous studies strongly suggest that stimulus-dependent protein lipidation is an essential part of the intricate signaling machinery controlling T cell activation and function. Therefore, we hypothesize that dynamic S-acylation of Orai1 and STIM1 is a critical regulator of calcium entry in T cells. To uncover the role of protein lipidation in calcium signaling, we will (1) determine whether Orai1 and STIM1 are S-acylated proteins in T cells, (2) determine the functional consequences of Orai1 and STIM1 acylation and (3) identify the enzymatic mechanisms mediating Orai1 and STIM1 S-acylation. The successful completion of the proposed project will demonstrate the biological significance of protein lipidation in regulation of SOCE as well as the role of palmitoyl acyltransferases in regulation of the calcium signaling in T cells with relevance to primary immunodeficiency disease.